Method for sterilization of biological preparations

ABSTRACT

Provided is a method for the sterilization of a biological preparation including desired viable biological entities. The method includes irradiating a dried (e.g. freeze-dried) biological preparation with UV radiation at an intensity and for a duration sufficient to reduce the amount or activity of living-matter contaminants in the biological preparation, the intensity and duration selected such that at least part of the desired biological entities in the sample remains viable. The described method is particularly suitable for the reduction of the amount or activity of contaminants such as bacteria or viruses from biological preparations including red blood cells or platelets.

FIELD OF THE INVENTION

The invention relates to the sterilization of biological preparations.More specifically the present invention relates to a method for thesterilization of biological preparations and to sterilized biologicalpreparations.

LIST OF REFERENCES

The following references are considered to be pertinent for the purposeof understanding the background of the present invention:

-   -   1. US 2004/067157 Methods for Sterilizing Biological Materials;    -   2. WO 2004/009138 Methods for Sterilizing Milk;    -   3. PCT IL2005/000125 Biological Material and Methods and        Solutions for Preservation Thereof;    -   4. U.S. Pat. No. 5,709,992 Method for disinfecting red blood        cells;    -   5. U.S. Pat. No. 6,482,585 Storage and maintenance of blood        products including red blood cells and platelets;    -   6. Hustom, et al. Lack of efficacy for conventional gamma        irradiation of platelet concentrates to abrogate bacterial        growth. Am J Clin Pathol. 1998; 109(6):743-7    -   7. Smith, et al. Gamma irradiation of HIV-1. J orthop Res. 2001;        19(5): 815-9.

BACKGROUND OF THE INVENTION

When storing cells, tissue or other biological material, there is alwaysthe danger of contamination from bacteria, viruses, yeasts, molds, fungietc., and sometimes the contaminants are present in the biologicalmaterial when it is first collected. Contaminants are such agents thatmay damage the biological material during preservation and/or harm therecipient when the product is used (e.g. transfused, injected or eaten).Among known contaminants are white blood cells (WBC) that are normallypresent in red blood cell (RBC) samples. The presence of WBC in atransfusion liquid is a problem due to graft vs. host disease, in whichthe transfused WBC (mainly the lymphocytes) attack the recipient's body.

Many methods for sterilization are known in the art including heatingand filtration. However, these processes may damage biological material(e.g. when it is sensitive to heat) or prove to be inefficient (e.g.when the biological material is filtered with some contaminants). Otherways for sterilization involve ionizing radiation, mainly gamma rays.For example, gamma radiation is used for inactivation of WBC, mainly thelymphocytes which are the main cause for graft versus host disease(GVHD), in fresh blood units. This is normally done by irradiating aliquid sample of blood or blood components including RBC, platelets,granulocyte components and non frozen plasma in a plastic bag with 2.5mega Rad of gamma radiation to the central portion of the bag, resultingwith no less than 1.5 mega Rad which are delivered to every part of theblood bag (AABB Technical Manual, 14^(th) edition). Attempts were madeto reduce the bacteria content in platelet concentrates (Hustom et al.1998) but it was concluded that exposure to gamma radiation at levels upto 7.5 mega Rad was ineffective at sterilizing the sample. Likewise itwas found that gamma radiation (1.5-2.5 mega Rad) does not constitute avirucidal dose for HIV type 1 in frozen bone and tendon allografts(Smith et al. 2001).

Furthermore, gamma radiation can be damaging to radiation-sensitiveproducts. In particular it has been shown that gamma radiation isinjurious to red blood cells, platelets and granulocytes (US2004/067157).

Ultraviolet (UV) radiation on the other hand is considered less damagingthan gamma radiation. However, as UV radiation is absorbed by water, itis practically ineffective for removal of contaminants that are in awater-containing sample (liquid or ice). Accordingly it was suggested inWO 2004/0091938 that reduction of the residual solvent content ofbiological material would reduce the absorption of UV in the water andthus enable sterilization of a biological sample using UV. However,sterilization of biological material in WO 2004/0091938 was restrictedto wet biological material or to non-cellular portions of a bloodpreparation (i.e. not including RBC or platelets), apparently since“sensitive biologicals, such as blood, would lose viability and activityif subjected to freezing for irradiation purposes and then thawing priorto administration to a patient” (id.).

GLOSSARY

The term “biological preparation” denotes a preparation or sample(natural, processed or man made) comprising desired biological entities.“Desired biological entities” are viable nucleus free biologicalentities, including eukaryotic nucleus free cells (e.g. RBC), parts ofcells (e.g. platelets), or artificial or semi-artificial material suchas liposomes. Examples of such biological preparations include blood orfractions thereof that contain RBC or platelets, an RBC-enrichedfraction of blood, packed RBC or platelet-enriched fraction of blood,samples of liposomes, etc.

A “Viable” biological preparation is such that at least a portion of thedesired biological entities therein appear to be structurally intact, orpreferably that they at least partially retain a desired biologicalactivity or if in a dry state may resume that activity upon rehydration.Preferably at least 10% of the desired biological entities are viable,desirably at least 30% or even at least 50%. In the case of RBC forexample a preferred percentage of viable cells may in some cases be atleast 75%.

In this invention, “liposomes” mean hollow lipid vesicles. They may beused to entrap the substance to be delivered within the liposomes, orthe drug molecule of interest can be incorporated into the lipid vesicleas an intrinsic membrane component, rather than entrapped into thehollow aqueous interior, or electrostatically attached to the aggregatesurface.

The term “living-matter contaminants” is taken to mean biologicalentities that contain genetic material and are therefore radiationsensitive. The living-matter contaminants may be present in a biologicalpreparation, either at the time of harvesting or may contaminate thebiological preparation at a later time (e.g. during its manipulation orstorage), and may damage the biological preparation or a portionthereof, its recipient or otherwise interfere with the use of thebiological preparation. Such living-matter contaminants may be any typeof biological entity including, nucleic acid sequences, prokaryotesincluding viruses, mycoplasma or bacteria and, fungi, yeasts, molds,single cell or larger parasitic microorganisms, or other undesiredcellular entities, such as WBC, etc.

The “Activity” of contaminants means any activity that may damage thebiological preparation, its recipient or otherwise interfere with theuse of the biological preparation (including due to legal constraints).The contaminants are of such radiation sensitivity that upon irradiationthey are reduced in number or activity, for example by becoming lesslikely to multiply (e.g. bacteria, WBC, yeast) or less likely to infecttarget cells (e.g. viruses) or transfect cells (nucleic acid sequences)or less likely to display a significant immune effect (e.g. WBC). Theamount or activity of the contaminants may be assayed, directly orindirectly, using any method of the art.

The term “ionizing radiation” means any form of radiation that hasenough energy to remove electrons from substances it passes through,forming ions. This includes alpha and beta particles, gamma radiationand x-rays. The term “UV radiation” means radiation having a wavelengthbetween 100-400 nm. It includes three ranges: UV-A (315-400 nm), UVB(280-315 nm) and UV-C (100-280 nm).

The terms “drying” “dried” or “dry” mean having (or causing to have) areduced water content as compared to the water content before drying. Adried preparation may have 10% less water than the original preparationfrom which it was derived, preferably less than 60% or even 75%, anddesirably 90% less water than the original preparation. Drying may bedone using any method known in the art, including air drying, heatdrying, freeze drying, spray drying or nebulizing, as long as thebiological preparation maintains viability of the desired biologicalentities. Examples of methods include air drying of liposomes (Hincha etal. 2003; Biochemica et Biophysica ACTA. 1612(2): 172-177), embryonickidney cell line and human foreskin fibroblasts cells (Gau et al. 2000;Nature Biotechnology. 18:168-171) etc. It is noted that bacteria maysurvive the air-drying process (Desmond et al. J. Appl Microbiol.2002;93(6):1003-11) and so can other contaminants.

The terms “lyophilization” or “freeze-drying” denote a process whereinmaterial is frozen and dried. Thus, in the present invention wherever abiological preparation is said to be freeze dried or lyophilized, thismay mean that at least two steps were executed, one of which forfreezing the sample and the other for drying. Each of these steps may bedone using any known method, and preferably such known methods thatcause minimal damage to the desired biological entities. Preferredmethods of freeze-drying are disclosed in PCT IL2005/000125, the contentof which is incorporated herein in full by way of reference.

SUMMARY OF THE INVENTION

The present invention is based on the inventors' surprising finding thatbiological preparations comprising desired nucleus free biologicalentities, such as RBC or platelets, may be irradiated using ionizing orUV radiation, when in a dried (e.g., freeze-dried) state such thatundesired living-matter contaminants will be destroyed with relativelylittle damage to said biological entities. “Little damage” should betaken to mean that at least 10% of the desired biological entities areviable, desirably at least 30% or even at least 50% of said biologicalentities is viable after irradiation. The invention is particularlysuitable for biological preparations that are freeze dried andrehydrated as described in PCT application PCT Il2005/000125, thecontents of which is incorporated herewith by reference in full, albeitnot limited thereto. The invention permits, according to an embodimentthereof, the irradiation and sterilization of biological preparationscomprising desired nucleus free biological entities using ionizing or UVirradiation.

Thus, the present invention provides according to one aspect a methodfor the sterilization of a biological preparation comprising desiredviable biological entities, the method comprising irradiating a driedbiological preparation with ionizing or UV radiation at an intensity andfor a duration sufficient to reduce the amount or activity ofliving-matter contaminants in the biological preparation, the intensityand duration selected such that at least part of the desired biologicalentities in the sample remains viable.

The present invention is particularly suitable for biologicalpreparations comprising desired biological entities derived from blood,including RBC and platelets.

DETAILED DESCRIPTION OF THE INVENTION

As detailed above, the present invention provides a method for thesterilization of biological preparations comprising desired viablebiological entities, allowing reduction of the amount or activity ofliving-matter contaminants in the biological preparation. Potentially,the amount of active contaminants in the biological preparation isreduced to none. In cases when a single biological preparation comprisesone or more contaminants before irradiation, it is intended that themethod of the present invention would allow the reduction of the amountor activity of at least one of said contaminants. Furthermore, in somecases, before irradiation the biological preparation might be free ofactive contaminants, in which case the present invention would ensurethe lack of contaminants and thus diminish, or even negate, the need tocheck for active contaminants.

According to some embodiments, the method also includes a step of dryinga biological preparation comprising desired viable biological entities.It is hence noted that the step of irradiating the biologicalpreparation may be performed at any time after the biologicalpreparation will become dry or partially dried. In fact, the irradiatingmay be done simultaneously (or partially simultaneously) with or even inbetween two steps of drying the biological preparation.

Any type of ionizing or UV radiation may be suitable for the presentinvention, however a person skilled in the art would appreciate that thetype, intensity and duration of irradiation would best be chosen so asto retain as much as possible the viability of the biologicalpreparation while reducing as much as possible the amount or activity ofcontaminants.

In order to understand the invention and to see how it may be carriedout in practice, preferred embodiments will now be described, by way ofnon-limiting example only.

Experiments Materials and Methods

Unless otherwise noted, all materials were purchased from Sigma Inc.(St. Louis. Mo., USA).

Example 1 The Effect of UV Exposure on the Survival of Lyophilized RBC

The effects of irradiation and freeze drying on red blood cells (RBC)were evaluated in this experiment. The freezing solution used wascomposed of 30% (w/v) dextran in PBS (Ca²⁺ and Mg²⁺ free). Packed RBCobtained from the Israeli Blood Services were mixed at a ratio of 1:1(v/v) with the freezing solution. 2.5 ml of RBC solution was put in a 16mm diameter of glass test tubes (Manara, Israel) which were then frozen.Freezing was done using the MTG freezing device (IMT, Israel) at acooling rate of 1000° C./min; (thermal gradient) G=5.5° C./mm, V=3mm/sec. The samples were also rotated at 56 RPM (rounds per minute).

After freezing, samples were put in a lyophilizer (Labconco, USA) for 3days (condenser −80° C.). After 3 days of lyophilization, when thesamples contained 10% or less of their original water content, onesample was placed in a Petri dish and exposed to UV radiation for 1 hourand the other was protected from light using aluminum foil. After 1 hourirradiation the samples were rehydrated with ultra pure water at 37° C.to their original volume. RBC were counted and hematocrit assayed usingthe Pentra 60 (ABX, France).

TABLE I Effect of UV exposure on lyophilized RBC Lyophilized RBC exposedto UV not exposed to UV Amount of cells 52.04% 57.6% hematocrit  29.2%35.5% The results are shown as a percentage of the fresh sample, beforefreezing.

As seen in Table I the sample that was exposed to UV exhibited aslightly lower survival rate than that of the sample that was notexposed to radiation. Since the inventors discovered that addition ofpolyphenols to the freezing solution improves the cells' survival infreeze-drying—thawing treatments, in the following experiments one suchpolyphenol was added to the biological samples. The term “polyphenols”denotes one or more natural and/or synthetic polyphenols that may benaturally found in green tea, including epigallocatechin gallate (EGCG),epicatechin gallate (ECG) epigallocatechin (EGC) epicatechin (EC).

Example 2 The Effect of UV Radiation on Lyophilized RBC Survival

In this experiment packed RBC were frozen with a freezing solutioncontaining: 30% (w/v) dextran 40,000 Dalton and 0.47 mg/ml EGCG (CaymanChemical, USA). The freezing solution and the packed RBC were mixed in aratio of 1:1 (v/v). 2.5 ml of the cell suspension were put in 16 mmdiameter glass test tubes (Manara, Israel). A total of 4 test tubes werefrozen. The samples were frozen at a cooling rate of 1000° C./min;(thermal gradient) G=5.5° C./mm, V=3 mm/sec using the MTG Device (IMT,Israel). The samples were also rotated at 56 RPM.

After freezing, samples were placed in liquid nitrogen. After thepassage of varying time periods (between ½ hour to a few weeks) sampleswere placed in a lyophilizer (Labconco, USA) with a condenser temp of−80° C.) for 72 hours, and the samples were dried such that they had theappearance of a powder and had less than 10% of their original watercontent. Then samples were transferred to a 60 mm Petri dish, 2 sampleswere exposed to UV for 1 hour and during that 1 hour the other 2 sampleswere covered with aluminum foil to prevent exposure to light. Allsamples were then rehydrated with ultra pure water at 37° C. to theiroriginal volume and compared using the PENTRA 60 counter (ABX, France).Results are presented as compared to the parameters of fresh RBC in afreezing solution including EGCG.

TABLE II Effect of UV radiation on lyophilized RBC survival LyophilizedRBC no UV treatment UV treatment Cells number 58.11% 54.05% Hematocrit43.02% 45.95% Results are shown as percentage of the fresh values of thesame samples

As can be seen from Table II, although more than 50% of the RBC appearedviable, freeze-dried cells were less viable and had a lower hematocritthan fresh cells. Nevertheless, these parameters were only slightlyaffected by UV radiation.

Example 3 The Effect of Partial Drying on RBC Survival

Fresh whole rat's blood (extracted from Sprague-Dawley rats) was washedonce. Plasma was removed and the packed RBCs were suspended in a 1:3ratio (v/v) with a freezing solution composed of 0.945 mg/ml EGCG and20% (w/v) Dextran 40 kD in 0.9% (w/v) NaCl solution, and the finalhematocrit was 25%. Three samples (2.5 ml each) were frozen each in a 16mm diameter glass test tube (Manara, Israel) using the MTG device (IMT,Israel), with the following parameters: velocity=3 mm/sec; temperaturegradient was 5.5° C./mm, the test tubes were rotated at 60 rpm. Afterfreezing, samples were stored in LN until lyophilization. Lyophilizationwas done in a special lyophilization device (IMT, Israel) subject ofco-pending PCT application No. IL2005/000124, which has a condensertemperature of −190° C. and samples were kept at a temperature of −20°C. Samples remained in the device for 48 hours. After 48 hours sampleswere taken out and thawed in a 37° C. water bath. Since, the sampleswere partly dried 1.5 ml 37° C. PBS (Ca²⁺ and Mg²⁺ Free) was added torehydrate the cells. PBS was added in stead of water since adding wateris expected to cause more damage to the cells than excess PBS.

The samples were then evaluated using the Pentra 60 cell counter (ABX,France) for a complete blood count evaluation, and supernatant freehemoglobin levels were measured using the cyanmethemoglobin method usingDrabkin's reagent. The absorbance was read at a wavelength of 540 nmusing a luminometer (Turner Biosystems, USA). The percentage of thesupernatant free hemoglobin (Hb) was calculated using the followingFormula I:

$\begin{matrix}{{\% \mspace{14mu} {Free}\mspace{14mu} {hemoglobin}} = {100 \times \frac{\left( {{Absorbance}\mspace{14mu} {of}\mspace{14mu} {the}\mspace{11mu} {supernatant}} \right)}{\begin{pmatrix}{{{Absorbance}\mspace{14mu} {of}\mspace{14mu} {supernatant}} +} \\{{Absorbance}\mspace{14mu} {of}\mspace{14mu} {the}\mspace{14mu} {pellet}}\end{pmatrix}}}} & {{Formula}\mspace{14mu} I}\end{matrix}$

TABLE III Partially dried RBC samples MCV Cell number Rat RBCs % Waterloss Free Hb (%) (%)* (%)* Fresh (3:1 ratio) — 5.89 — Lyophilized 60%22.26 ± 0.16 76.60 78.63 *Results given as percentages of thawed valuesas compared to fresh values.

Lyophilization for 48 hours resulted in about 60% of water loss. Thiswater loss was evaluated by the amount of PBS that was needed to beadded to the solution in order to regain the original sample's volume.In the freeze dried sample there was some cell damage as seen in thefree hemoglobin percentage (22.26% Free Hb). However, microscopicobservations revealed more than 50% of the cells with normal morphology.In addition, this free hemoglobin rate might be a result of the thawingprocess, since upon thawing and before addition of PBS the thawed cellswere exposed to a very hypertonic environment, which remained hypertonicbut to a lesser extent after PBS was added.

Example 4 The Effect of Freezing and Freeze-Drying on E. coli

E. coli were placed in LB medium: 10 gr Bacto-tryptone (Difco, USA), 5gr yeast extract (Difco, USA), 10 gr NaCl, in 1 liter distilled water.The total volume of 10 ml was divided to two batches of 5 ml each. Tothe first batch of E. coli in LB medium we added 5 ml of freezingsolution composed of 30% (w/v) dextran and 0.47 mg/ml EGCG (CaymanChemical, USA) in PBS (Ca⁺² and Mg⁺² free). The other batch was leftun-touched. Cell-suspension samples of 2.5 ml each (two from each batch)were put in 16 mm diameter glass test tubes (Manara, Israel), such thata total of 4 test tubes were prepared. The test tubes were frozen usingthe MTG Device (IMT, Israel) at 1000° C./min (from 5 to −50° C. at avelocity of 3 mm/sec and with 56 RPM. After freezing was completed thetest tubes were placed in liquid nitrogen. Afterwards, the 4 test tubeswere placed in a lyophilizer (Labcono, USA) for 72 hours. Afterlyophilization was completed the “powdered” cells from each test tubewere scraped into a Petri dish. Two Petri dishes (one representing eachbatch) were exposed to UV radiation for 1 hour (the Petri dishes wereplaced opened under a UV lamp) and the other two Petri dishes were leftunexposed to radiation (covered with aluminum foil for protection fromlight). After 1 hour 2 ml of double distilled water at 37° C. were addedto each dish. From each dish 3 Petri dishes with agar were plated. Thefollowing Agar plates protocol was used: 10 gr Bacto-tryptone, 5 gryeast extract, 10 gr Na⁺ Cl⁻, 10 gr agar (BD, USA). Water was added to avolume of 1 liter, autoclaved, cooled to 65° C. and poured into Petridishes. A total of 12 Petri dishes were incubated at 37° C. for 24hours. The next day colonies were counted. Table IV depicts the numberof colonies grown on the agar Petri dishes.

TABLE IV Number of E. coli colonies after being frozen with differentfreezing solutions and lyophilized E. coli E. coli frozen frozen withdextran in LB and EGCG — UV — UV 36 0 152 0 24 0 220 0 16 0 >200 0

As seen in Table IV, E coli colonies were observed only in the plates ofthe un-irradiated bacteria. No colonies were observed in the agar platedwith lyophilized cells that were irradiated. In addition, the additionof Dextran and EGCG results in higher survival rates of the bacteriaafter lyophilization.

Example 5 The Effect of Freezing and Freeze-Drying on E. coli in an RBCPreparation

10 ml of E. coli in LB medium was centrifuged at 800 g for 10 minutes.To the resultant pellet 10 ml of freezing solution composed of 30% (w/v)dextran 40,000 Dalton and 0.47 mg/ml EGCG (Cayman Chemical, USA) in PBS(Ca⁺² and Mg⁺² free) were added. This solution was then mixed in avolumetric ratio of 1:1 with packed RBC. 2 ml of RBC & E. coli were putin a Petri dish; a total of 4 like dishes were prepared. 2 Petri disheswere exposed to UV for 1 hour and the other 2 were not. After 1 hourcells from each group were plated on three agar plates that were placedin a 37° C. oven for 24 hours.

From the remaining RBC-coli mixture four test tubes were prepared, eachcontaining 2.5 ml. The test tubes were frozen using the MTG device (IMT,Israel) at 1000° C./min (from 5 to −50° C. at a velocity of 3 mm/sec andwith 56 RPM and then placed in a lyophilizer for 72 h. Afterlyophilization one test tube from each group was exposed to UV radiationfor 1 hour. After 1 hour 2 ml of ddH₂O was added and from each group 3agar plates were seeded and placed for 24 hour in 37° C. oven for 24hours. The results are depicted in Table IV.

TABLE V The effect of UV radiation on the survival of E coli inlyophilized or fresh samples comprising RBC Fresh RBC Lyophilized andRBC and E. coli E. coli — UV — UV >200 >200 >200 0 >200 >200 >2000 >200 >200 >200 0

As seen in Table V irradiation in the liquid state had no measuredeffect on E. coli, as in all plates more then 200 colonies wereobserved. However, when irradiated in a dry (lyophilized) state nocolonies were observed after 24 hours in incubation.

Example 6 The Effect of UV Radiation on the Survival of E. coli in FreshPlatelets Concentrates

A unit of fresh platelets was received from the Israeli blood bank.Platelets were added to an E. coli pellet (E. coli in LB medium that wascentrifuged for 10 minutes at 2000 g). The platelets & E. coli solutionwas mixed at a ratio of 1:1 (v/v) with a freezing solution composed of30% (w/v) Dextran (40,000 Dalton; Amersham Biosciences, USA) and 1.87mg/ml EGCG (Cayman, USA) in PBS (calcium and magnesium free). Twosamples, 2.5 ml each, of platelet suspension were put in a 60 mm Petridish. One dish was exposed to UV radiation for 1 hour, and the other wasleft untouched, covered in aluminum foil. After one hour, samples fromeach Petri dish were seeded in agarose and put in an incubator at 37° C.for 24 hours. After 24 hours colonies were counted.

TABLE VI The effect of UV radiation on the number of E. coli coloniesExposed to UV Not exposed to radiation 269 201

We can see that UV irradiation of E. coli in a fresh plateletconcentrate did not have an effect on the E. coli survival, resulting in269 colonies in the sample that was exposed to radiation and in 201 inthe sample that were not exposed to UV radiation.

Example 7 The Effect of UV Radiation on the Number of E. coli ColoniesGrown after being Lyophilized

Platelets-E. coli solutions were prepared as described in Example 6. Theplatelets-E. coli solution was divided to two batches, and each batchwas mixed at a ratio of 1:1 (v/v) with one of the following freezingsolutions: (1) 30% (w/v) Dextran (40 KDa) and 1.87 mg/ml EGCG in PBS(calcium and magnesium free); or (2) 30% (w/v) Dextran (40 KDa) in PBS(calcium and magnesium free). 2.5 ml aliquots of platelet suspensionwere put in 16 mm diameter glass test tubes (Manara, Israel). A total of4 test tubes were prepared, 2 tubes from each batch. The tubes werefrozen in the MTG device at a thermal gradient of 5.5° C./mm and at acooling rate of 1000° C./min (final temperature was −50° C., velocitywas 3 mm/sec).

After freezing, all tubes were maintained in liquid nitrogen and laterlyophilized for 3 days, such that the preparation appeared as a powdercontaining less than 10% of its original water content. The resultantdry powder was scraped into a 60 mm Petri dish, such that two disheswere prepared from each of the above batches. One dish from each batchwas exposed to UV radiation for 1 hour. The other 2 dishes (one fromeach batch) were untouched, covered in aluminum foil. The contents ofeach Petri dish were rehydrated with 2 ml of ultra pure water at 37° C.and a sample from each dish was seeded in agarose and incubated at 37°C. for 24 hours. After 24 hours colonies were counted.

TABLE VII The effect of UV radiation on the number of E. coli coloniesgrown after being lyophilized batch UV No UV Platelets - E. coli &Dextran 30% 1 17 Platelets - E. coli & Dextran + EGCG 1 11

As seen in Table VII, UV radiation reduced the number of colonies bymore than tenfold.

In order to assess the platelets' survival of the UV irradiation in adry state, samples of platelets (prepared with EGCG and Dextran asdescribed above) taken after lyophilization and rehydration werecompared with those taken after UV irradiation. The platelets werecounted using the Pentra 60 (ABX, France) cell counter, and it wasobserved that 80.38% of the platelets that survived lyophilization alsosurvived UV treatment.

Example 8 Sterilization by Gamma Radiation of Lyophilized RBC SamplesContaminated with West Nile Virus (WNV) Example 8A Sterilization of RBCusing Gamma Radiation

Packed RBC that were received from the Israeli Blood Services were mixedin a volumetric ratio of 1:1 with a freezing solution composed of 20%(w/v) Dextran 40 kD and 0.945 mg/ml EGCG and 0.9% (w/v) NaCl in doubledistilled water. 2.5 ml samples were contaminated with WNV (receivedfrom the Israeli Veterinary Institute) to the following virusconcentrations: 10^(6.8) WNV/ml blood (referred to Max), 10^(5.8) WNV/mlblood (referred to as −1) and 10^(4.8) WNV/ml blood (referred to as −2).Uncontaminated blood was used as a control for the infection.

The 2.5 ml samples were frozen using the MTG freezing device (IMTIsrael), in the same conditions as described above. After freezing wascompleted samples were stored in liquid nitrogen until put in alyophilizer (condenser temperature −80° C.) (Labconco, USA) for 72hours.

The freeze-dried blood was exposed to gamma radiation of one of threeintensities (1, 2.5 and 5 mega Rad), whilst a control for theirradiation was kept without irradiation. After the irradiation allsamples were rehydrated with double distilled water at 37° C. to thesamples' original volume.

Survival of the viruses was assayed by injection of 0.03 ml bloodsamples to the brain of newborn mice. The mice were monitored for up to14 days after infection, during which the number of mice that died afterdisplaying WNV symptoms was recorded. The results are summarized inTable VIII

TABLE VIII Mice mortality due to injection of Lyophilized RBCs whichwere contaminated with WNV Gamma radiation Virus amount number conc.(Mega Rad) of mice mortality max 0 11 11 −1 0 11 11 −2 0 11 11 none 0 110 max 1 10 10 −1 1 10 0 −2 1 10 0 none 1 10 0 max 2.5 11 0 −1 2.5 11 0−2 2.5 11 0 none 2.5 11 0 max 5 11 0 −1 5 11 0 −2 5 11 0 none 5 11 0

As can be seen in Table VIII above, irradiation of freeze dried RBC bygamma radiation has significantly reduced the activity of WNV in all theexperimented levels of radiation. Even at the intensity of 1 mega Radthe radiation has reduced the WNV activity in the lower concentrationsof −1 and −2 below a detectable level. This level of radiation (1 megaRad) is well bellow what is commonly used for WBC inactivation of bloodsamples.

Those skilled in the art will readily appreciate that variousmodifications and changes can be applied to the embodiments of theinvention as hereinbefore described without departing from its scopedefined in and by the appended claims and their equivalents. Also, it isto be understood that the phraseology and terminology employed hereinare for the purpose of description and should not be regarded aslimiting.

1.-6. (canceled)
 7. A method for sterilizing a biological preparationcomprising a viable eukaryotic nucleus free cell, the method comprising:irradiating a freeze-dried biological preparation comprising a viableeukaryotic nucleus free cell with UV radiation at an intensity and for aduration sufficient to reduce the amount or activity of living-mattercontaminants in the freeze-dried biological preparation, the intensityand duration selected such that at least part of the viable biologicalentity in the freeze-dried biological preparation remains viable,wherein the freeze-dried biological preparation does not comprise asensitizing agent and the method does not comprise adding a sensitizingagent.
 8. The method of claim 7, wherein the UV radiation is irradiatedfor a period of 1 hour or less. 9-11. (canceled)
 12. The method of claim7, wherein the freeze-dried biological preparation has less than 60%less water than the original preparation.
 13. The method of claim 12,wherein the freeze-dried biological preparation has less than 90% lesswater than the original preparation.
 14. A viable biological preparationobtainable by the method of claim
 7. 15-16. (canceled)